Method for making reservoir-based sensor device

ABSTRACT

Methods are provided for making a reservoir-based sensor device. The method may include fabricating a first substrate portion which comprises an upper surface, an opposed lower surface, a plurality of through holes defining reservoirs between said upper surface and said lower surface, and a plurality of reservoir caps closing off one end of said through holes at said upper surface; fabricating a plurality of sensors on an upper surface of a second substrate portion; aligning the first and second substrate portions such that each of said plurality of sensors is in alignment with each of said plurality of reservoirs; and bonding together the lower surface of said first substrate portion and the upper surface of said second substrate portion to seal each of said sensors inside the reservoir with which it is aligned.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 11/850,687, filedSep. 6, 2007, now pending, which is a continuation of U.S. applicationSer. No. 11/039,048, filed Jan. 19, 2005, now abandoned, which is acontinuation of U.S. application Ser. No. 10/324,556, filed Dec. 19,2002, now U.S. Pat. No. 6,849,463, which is a continuation of U.S.application Ser. No. 09/798,562, filed Mar. 2, 2001, now U.S. Pat. No.6,551,838, which claims benefit of U.S. Provisional Application No.60/186,545, filed Mar. 2, 2000. These applications are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

This invention is in the field of miniaturized devices having reservoirswhich contain small devices or device components and/or chemicals.

Microarray systems have been developed that analyze numerous compounds,such as for drug activity or hybridization analysis of nucleotidemolecule sequences. For example, U.S. Pat. No. 5,843,767 to Beattiediscloses a microfabricated, flowthrough “genosensors” for the discretedetection of binding reactions. The apparatus includes a nanoporousglass wafer having tapered wells in which nucleic acid recognitionelements are immobilized. U.S. Pat. No. 6,083,763 to Balch discloses anapparatus for analyzing molecular structures within a sample substanceusing an array having a plurality of test sites upon which the samplesubstance is applied. The test sites typically are in microplate arrays,such as microtitre plates. These apparatuses, however, do not provideany means for sealing one or more of the wells or for selectivelyexposing one or more of the wells, for example, on demand or uponpassive exposure to certain conditions.

U.S. Pat. Nos. 5,797,898 and 6,123,861 to Santini, et al. describemicrochip devices that release drug molecules from reservoirs havingreservoir caps that actively or passively disintegrate. It would beadvantageous to adapt these devices for use in sensing applications andfor use in initiating or measuring chemical reactions in a micro-scalearea or volume at specific points in time.

U.S. Pat. No. 5,252,294 to Kroy discloses micromechanical structureshaving closed cavities for use in storage and handling of substances,for example, in research and testing of the substances. There is nodisclosure, however, of selectively controlling exposure of individualcavities without microvalves, nor is there any disclosure of isolatingindividual sensing means.

It would be desirable to provide miniaturized devices for use ininitiating and controlling chemical reactions, analyses, or measurementsin a micro-scale area or volume, at specific points in time. It wouldalso be desirable to provide methods of making and using suchminiaturized devices.

SUMMARY OF THE INVENTION

Microchip devices are provided to store and protect chemicals andsmaller, secondary devices from environmental exposure until such timeas exposure is required, for example, to initiate a chemical reactionand/or to perform an analysis or sensing function. In one embodiment,the microchip device includes a substrate having a plurality ofreservoirs which contain the secondary device, and at least one barrierlayer covering each reservoir to isolate the secondary device from oneor more environmental components outside the reservoirs. The barrierlayer can be selectively disintegrated or permeabilized to expose thesecondary device to the one or more environmental components. Thesecondary device preferably includes a sensor or sensing component, forexample, a biosensor, or a light detection or imaging device, such as anoptical fiber. In one variation, the microchip device further includes areacting component, such as catalyst or reagent, in one or morereservoirs. Alternatively, the sensor or sensing component can beattached to the substrate outside of the reservoir while a reservoircontains a reacting component.

In another embodiment, the microchip device includes a substrate havinga plurality of reservoirs which contain a reacting component, and atleast one barrier layer covering each reservoir to isolate the reactingcomponent from one or more environmental components outside thereservoirs. The barrier layer can be selectively disintegrated orpermeabilized to expose the reacting component to the one or moreenvironmental components. In a preferred variation, the reactingcomponent is a catalyst or enzyme that remains immobilized in thereservoir even after exposure to the environmental components. In someembodiments, swellable materials and osmotic pressure generatingmaterials can be incorporated into reservoirs for use in generatingpressure forces effective to rupture the barrier layer.

The microchip device is used to protect chemicals and devices fromexposure to the surrounding environment until the exposure is desired,which is particularly useful when the chemicals or devices within thereservoir are sensitive to environmental conditions, for example, whenthe devices fail or materials foul following prolonged exposure to theenvironment. In one embodiment, an easily fouled catalyst used toinitiate a desired heterogeneous chemical reaction is sealed inside areservoir of a microchip device to protect it from the surroundingenvironment. When it is desired to initiate the reaction, the barrierlayer on the reservoir is removed or made permeable. The reagents forthe reaction present in the surrounding environment pass into thereservoir (e.g., by diffusion), contact the catalyst, react at thecatalyst surface, and the products pass out of the reservoir. Thisheterogeneous reaction continues until the reagents are exhausted or thecatalyst becomes fouled. This process may be repeated numerous times byopening additional reservoirs and exposing fresh catalyst.

In another embodiment, the microchip device includes one or more sensorsthat are located inside each reservoir. The sensors are protected fromthe environment until the barrier layer is removed or made permeable.Once the barrier is removed or made permeable, the sensors can detectthe presence and/or quantity of molecules or the conditions in or nearone or more reservoirs. Such sensors can be used, for example, incontrolling and monitoring the release of molecules from other chemicalrelease devices or the release of chemicals from reservoirs in the samedevice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are cross-sectional diagrams showing various embodiments of asingle reservoir of the device, having a barrier layer over thereservoir and on top of the substrate (FIG. 1A), a barrier layer withinthe opening of the reservoir (FIG. 1B), and a combination thereof in adevice having two substrate portions bonded together (FIG. 1C).

FIGS. 2A-C are cross-sectional diagrams showing a device having areservoir covered by a semi-permeable barrier layer which permitspassage of molecule A into or out of the reservoir (FIG. 2A), whereinthe reservoir initially is covered by another, impermeable barrier layer(FIG. 2B) until the barrier layer is selectively removed (FIG. 2C).

FIGS. 3A-B are cross-sectional diagrams of two embodiments of an opticalsensing device, wherein the device includes an optical fiber inside(FIG. 3A) or outside and over (FIG. 3B) a single reservoir covered by asemi-permeable barrier layer which permits passage of molecule A into orout of the reservoir.

FIGS. 4A-B are cross-sectional diagrams illustrating a device having areservoir containing both a sensor and a chemical reagent, with anintact (FIG. 4A) and partially removed (FIG. 4B) impermeable barrierlayer.

FIGS. 5A-B are cross-sectional diagrams illustrating a device having onereservoir which contains a sensor and another reservoir which contains achemical reagent, with an intact (FIG. 5A) and partially removed (FIG.5B) impermeable barrier layers over the reservoirs.

FIG. 6 is a cross-sectional diagram illustrating a device having areservoir containing a chemical reagent and a sensor located on thesubstrate outside of the reservoir.

FIG. 7 is a process flow diagram illustrating one embodiment forcontrolling and communicating with sensors located in reservoirs of amicrochip device.

FIGS. 8A-B are cross-sectional diagrams illustrating a device having areservoir containing a catalytic material, with the intact barrier layerimpermeable to reactant A (FIG. 8A) and the barrier layer partiallyremoved and permitting reactant A to contact the catalytic material toyield product B (FIG. 8B). FIGS. 8C and 8D are diagrams illustratingoptional elements which can be incorporated into the reservoirs tocontrol the catalytic reactions, such as resistive heaters (FIG. 8C) andpolarizable electrodes (FIG. 8D).

FIGS. 9A-B are cross-sectional diagrams illustrating a device having areservoir containing an immobilized enzyme, with the intact barrierlayer impermeable to reactant A (FIG. 9A) and the barrier layerpartially removed and permitting reactant A to contact the immobilizedenzyme to yield product B (FIG. 9B).

FIG. 10 is a cross-sectional diagram of one embodiment of a deviceutilizing osmotic pressure forces generated within a first reservoir torupture a barrier layer covering a second, nearby reservoir.

FIGS. 11A-B are cross-sectional diagrams illustrating a device having abarrier layer covering a reservoir that contains both an osmoticpressure generating material and a chemical to be released (FIG. 11A),and the rupture of the barrier layer due to a pressure differentialproduced by osmotic forces (FIG. 11B).

FIGS. 12A-D are cross-sectional diagrams showing reservoirs havingcomplex reservoir shapes, which can be made, for example, by utilizingwafer or other substrate bonding methods or by adaptingsilicon-on-insulator (SOI) fabrication methods.

DETAILED DESCRIPTION OF THE INVENTION

Microchip devices are provided that store and protect reactingcomponents and secondary devices from the environment for a period oftime until exposure to the environment is desired. The microchipdevices, which provide for the selective or controlled exposure of thesecontents, include a plurality of reservoirs, the contents of which arecompletely or partially isolated until it is desired to expose areacting component or secondary device in the reservoir to theenvironment, or a portion thereof outside of the reservoir. The devicesare designed to restrict, enhance, or otherwise control the passage ofmolecules or energy into (or out of) the reservoirs. These functions areaccomplished by covering at least one opening of each reservoir of themicrochip device by at least one barrier layer.

As used herein, a “microchip” is a miniaturized device fabricated usingmethods commonly applied to the manufacture of integrated circuits andMEMS (MicroElectroMechanical Systems) such as ultraviolet (UV)photolithography, reactive ion etching, and electron beam evaporation,as described, for example, by Wolf & Tauber, Silicon Processing for theVLSI Era, Volume 1—Process Technology (Lattice Press, Sunset Beach,Calif., 1986); and Jaeger, Introduction to Microelectronic Fabrication,Vol. V in The Modular Series on Solid State Devices (Addison-Wesley,Reading, Mass., 1988), as well as MEMS methods that are not standard inmaking computer microchips, including those described, for example, inMadou, Fundamentals of Microfabrication (CRC Press 1997) andmicromolding and micromachining techniques known in the art. Themicrochip fabrication procedure allows the manufacture of devices withprimary dimensions (length of a side if square or rectangular, ordiameter if circular) ranging from less than a millimeter to severalcentimeters. A typical device thickness is 500 μm. However, thethickness of the device can vary from approximately 10 μm to severalmillimeters, depending on the device's application. Total devicethickness and reservoir volume can also be increased by bonding orattaching additional silicon wafers or other substrate materials to thefabricated microchip device. In general, changing the device thicknessaffects the volume of each reservoir and may affect the maximum numberof reservoirs that may be incorporated onto a microchip. In vivoapplications of the device typically require devices having a primarydimension of 3 cm or smaller for subcutaneous implantation, but may beup to several centimeters for peritoneal or cranial implantation.Devices for in vivo applications are preferably small enough to beswallowed or implanted using minimally invasive procedures. Smaller invivo devices (on the order of a millimeter) can be implanted using acatheter or other injection means. Microchip devices that remain outsideof the body, but that are used in a system for in vivo applications(e.g., sensing following extraction of a sample of biological fluid),have much fewer size restrictions. Devices for in vitro applicationsalso have fewer size restrictions and, if necessary, can be made muchlarger than the dimension ranges for in vivo devices.

I. Device Components and Materials

Each microchip device includes a substrate having reservoirs containinga reacting component or secondary device, wherein at least one openingof each reservoir is covered by a barrier layer that protects thecontents from one or more components of the surrounding environment.Examples of these environmental components include chemicals, water,biological fluids, cells, molecules, and one or more forms of energy,such as light or heat.

FIGS. 1A-C illustrate a cross-sectional view of various embodiments ofmicrochip device 10 comprising substrate 12, reservoir 14, backing plate16, and barrier layer 18. In the embodiment of FIG. 1C, the substrate iscomposed of substrate portions 12 a and 12 b, and the microchip devicefurther includes semi-permeable barrier layer 20. It should be notedthat the backing plate is typically utilized only in device embodimentsproduced by a process in which reservoirs are formed from a hole thatpasses completely through the substrate. The backing plate essentiallycan be any impermeable plate or layer of rigid or flexible material thatserves the sealing function.

The microchip devices may be classified as passive devices, in which thepermeability of the barrier layer changes without any user intervention,or active devices, in which the device controller initiates an actionwhich renders the barrier layer permeable. Active devices may includecontrol circuitry, memory, and a power source, and may be operable usingwireless or remote communication, control, and data and powertransmission.

A. The Substrate

The substrate contains the reservoirs and serves as the support for themicrochip. Any material which can serve as a support, which is suitablefor etching or machining or which can be cast or molded, and which isimpermeable to the contents of the reservoir and to the surroundingenvironment (e.g., water, blood, electrolytes, other solutions, or air)may be used as a substrate. Examples of suitable substrate materialsinclude ceramics, glasses, certain metals, semiconductors, anddegradable and non-degradable polymers. Biocompatibility of thesubstrate material is preferred, but not required. For in vivoapplications, non-biocompatible materials may be encapsulated in abiocompatible material, such as poly(ethylene glycol) orpolytetrafluoroethylene-like materials, before use. A few examples ofstrong, non-degradable, easily etched substrates that are impermeable tothe molecules or secondary devices contained in the reservoirs and tothe surrounding fluids are silicon, glass, and titanium. In anotherembodiment, the substrate is made of a strong material that degrades ordissolves over a period of time into biocompatible components. Thisembodiment is preferred for in vivo applications where the device isimplanted and physical removal of the device at a later time is notfeasible or is difficult, for example, brain implants. An example of aclass of strong, biocompatible materials is thepoly(anhydride-co-imides) described in Uhrich et al., “Synthesis andcharacterization of degradable poly(anhydride-co-imides)”,Macromolecules, 28:2184-93 (1995).

The substrate can be formed of only one material or can be a compositeor multi-laminate material, e.g., two or more substrate portions can bebonded together (see FIGS. 12A-C, described below). Multi-portionsubstrates can be formed of the same or different materials, includingfor example, silicon, glasses, ceramics, semiconductors, metals, andpolymers. Two or more complete microchip devices also can be bondedtogether to form multi-portion substrate devices (see FIG. 12D).

B. Secondary Devices and Reacting Components

The reservoirs contain secondary devices, reacting components, orcombinations thereof, that need to be protected from surroundingenvironmental components until their exposure is desired.

Secondary Devices

As used herein, unless explicitly indicated otherwise, the term“secondary device” includes, but is not limited to, any device andcomponent thereof which can be located in or designed to operablycommunicate with one or more reservoirs in a microchip device. In apreferred embodiment, the secondary device is a sensor or sensingcomponent. As used herein, a “sensing component” includes, but is notlimited to, a component utilized in measuring or analyzing the presence,absence, or change in a chemical or ionic species, energy, or one ormore physical properties (e.g., pH, pressure) at a site.

The secondary device can be integrated within each reservoir or placedin close proximity to the reservoirs. Secondary devices may comprise acomplete device or system or may be one component of a larger or morecomplex device. In one embodiment, a sensor present inside a reservoirremains isolated from the surrounding environment until the permeabilityof the reservoir's barrier layer is altered. When it is desired to usethe sensor, the barrier layer is removed or made permeable. Themolecules to be detected that are present in the surrounding environmentdiffuse into the reservoir and interact with the sensor. In anotherembodiment, a light detection or imaging device (e.g., optical cell, CCDchip, etc.) is located in a sealed reservoir until it is desired todetect an optical signal or capture an image. The barrier is removed ormade permeable so that light energy can pass through to the opticaldevice located in the reservoir.

Microchip devices also can store and expose any combination of chemicalsand devices. For example, each reservoir can contain a differentchemical or molecule for release. In one embodiment, devices can beplaced outside of, but in close proximity to several chemical releasereservoirs, in order to monitor when a chemical is released from aparticular reservoir. In another embodiment, the chemical contained inthe reservoir is an enzyme catalyst, glucose oxidase, which is used insome glucose sensing devices. It is also understood that multipledevices having completely different functions can be placed inside ornear each reservoir of a microchip device. For example, in oneembodiment, three sensors for detecting and quantifying three moleculescan be located in the same reservoir, while three completely differentsensors for detecting three different molecules can be placed in aneighboring reservoir. Alternatively, a single device may be comprisedof three components, each of which is located in a different reservoir.With this technology, a microchip has the ability to selectively exposeeach chemical, device, or device component to the environment outside ofthe reservoir and to vary the number and type of chemicals and devicesassociated with each reservoir.

In a preferred embodiment, the secondary device is a sensor. Types ofsensors that can be contained within or provided near a reservoirinclude biosensors, chemical sensors, physical sensors, or opticalsensors. Preferred sensors measure properties such as biologicalactivity, chemical activity, pH, temperature, pressure, opticalproperties, radioactivity, and electrical conductivity. These may bediscrete sensors (e.g., “off-the-shelf” sensors) or sensors integratedinto the substrate. Biosensors typically include a recognition elementsuch as an enzyme or antibody. The transducer used to convert theinteraction between the analyte and recognition element into anelectronic signal may be, for example, electrochemical, optical,piezoelectric, or thermal in nature. Representative examples ofbiosensors constructed using microfabrication methods are described inU.S. Pat. Nos. 5,200,051; 5,466,575; 5,837,446; and 5,466,575 toCozzette, et al.

The sensor may be one for detecting carbon dioxide, carbon monoxide,ammonia, dioxygen, ethanol, ionized calcium, sodium ion, potassium ion,lithium ion, hydrogen ion, chloride ion, magnesium ion, ammonium ion,hydrogen peroxide, ascorbic acid, cholesterol, uric acid, esterifiedcholesterol, urea, creatinine, creatine, triglycerides, lactatedehydrogenase, creatine kinase, alkaline phosphatase, creatinekinase-MB, alanine transaminase, aspartate transaminase, bilirubin,amylase, or lipase.

In one embodiment, the sensor comprises an immunoassay. The sensor mayinclude an electrochemical transducer. The transducer may beamperometric, potentiometric, or conductimetric.

In one embodiment, the sensor may include at least one electrode, anadhesion promoter layer on the at least one electrode, and a bioactivelayer that is immobilized on the adhesion promoter layer. The bioactivelayer may include an enzyme selected from glucose oxidase, glucosedehydrogenase, NADH oxidase, uricase, urease, creatininase, sarcosineoxidase, creatinase, creatine kinase, creatine amidohydrolase,cholesterol esterase, cholesterol oxidase, glycerol kinase, hexokinase,glycerol-3-phosphate oxidase, lactate dehydrogenase, alkalinephosphatase, alanine transaminase, aspartate transaminase, amylase,lipase, esterase, gamma-glutamyl transpeptidase, L-glutamate oxidase,pyruvate oxidase, diaphorase, bilirubin oxidase, and mixtures thereof.

There are several different options for receiving and analyzing dataobtained with devices located in the microchip devices. First, theoutput signal from the device can be recorded and stored in writeablecomputer memory chips. Second, the output signal from the device can bedirected to a microprocessor for immediate analysis and processing.Third, the signal can be sent to a remote location away from themicrochip. For example, a microchip can be integrated with a radiotransmitter in order to transmit a signal (e.g., data) from themicrochip to a computer or other remote receiver source. The microchipcan also be controlled using the same transmission mechanism. Power canbe supplied to the microchip locally by a microbattery or remotely bywireless transmission.

Reacting Components

As used herein, unless explicitly indicated otherwise, the term“reacting component” includes any chemical species which can be involvedin a reaction, including, but not limited to, reagents; catalysts,including enzymes, metals, and zeolites; proteins; nucleic acids;polysaccharides; polymers; cells, as well as organic or inorganicmolecules, including diagnostic agents.

The reacting component contained within a reservoir may be present inany form (e.g., solid, liquid, gel, or vapor). They may be present inthe reservoir in pure form or as a mixture with other materials. Forexample, the chemicals may be in the form of solid mixtures, such asamorphous and crystalline mixed powders, porous or nonporous monolithicsolid mixtures, and solid interpenetrating networks; liquid mixtures orsolutions, including emulsions, colloidal suspensions, and slurries; andgel mixtures, such as hydrogels. When the barrier layer is removed froma reservoir, the chemicals inside the reservoir can remain in thereservoir or can be released from the reservoir.

In one embodiment wherein the chemicals remain in the reservoir, thechemicals are zeolites used for a heterogeneous reaction. When thebarrier layer is removed, the reagents diffuse into the reservoir toreact at the surface of the zeolite catalyst, which remains in thereservoir. In one embodiment wherein the chemicals are released from thereservoir, molecules originally contained in the reservoir are releasedfrom the reservoir in vitro where the controlled release of a small(milligram to nanogram) amount of one or more molecules in a particularsequence is desired, for example, in the fields of analytic chemistry ormedical diagnostics. Chemicals released in such a way can be effectiveas pH buffering agents, diagnostic agents, and reagents in complexreactions such as the polymerase chain reaction or other nucleic acidamplification procedures.

D. Barrier Layer

At least one opening of each reservoir of the microchip device iscovered by a barrier layer, which separates (i.e. isolates) the contentsof the reservoir from the surrounding environment or from portionsthereof. The barrier layer can be impermeable, permeable, orsemi-permeable to molecules or energy (e.g., light or electric field).The permeability of the barrier layer to molecules or energy can beactively controlled by the selective, real-time removal of all or partof the barrier layer by, for example, an applied stimulus (e.g.,electric field or current, magnetic field, change in pH, or by thermal,photochemical, chemical, electrochemical, or mechanical means) or can bepassively controlled by the barrier layer's structure, composition, ormethod of fabrication. For example, the passage of molecules or energyinto each reservoir of a device can be controlled by diffusion (e.g.,through a solid cap material, a nanoporous material, or a microporousmaterial), osmotic pressure, ionic gradients, electric fields orcurrents, capillary forces, or surface tension.

The barrier layer can multi-layered. It can include a membrane, areservoir cap, a plug, a thick or thin solid or semi-solid film, atwo-phase interface (i.e. solid-liquid, liquid-liquid, or liquid-gas),or any other physical or chemical structure suitable for separating thecontents of a reservoir from the environment outside of the reservoir.It generally is self-supporting across the reservoir opening.Selectively removing the barrier layer or making it permeable will then“expose” the contents of the reservoir to the environment (or selectedcomponents thereof) surrounding the reservoir.

In preferred embodiments, the barrier layer can be selectivelydisintegrated or permeabilized. As used herein, the term “disintegrate”is used broadly to include without limitation degrading, dissolving,rupturing, fracturing or some other form of mechanical failure, as wellas a loss of structural integrity due to a chemical reaction or phasechange, e.g., melting, in response to a change in temperature, unless aspecific one of these mechanisms is indicated. As used herein, the term“permeabilization” includes without limitation any means of renderingthe barrier layer porous or permeable in an amount effective to permitone or more species of molecules or forms of energy to pass in eitherdirection through the barrier layer. Puncturing of the barrier layer,such as by injecting a needle through the barrier layer into thereservoir, generally is not a preferred means of permeabilizing ordisintegrating the barrier layer.

In passive devices, the barrier layer is formed from a material ormixture of materials that degrade, dissolve, or disintegrate over time,or do not degrade, dissolve, or disintegrate, but are permeable orbecome permeable to molecules or energy. Barrier layer materials forpassive microchips are preferably polymeric materials, but barrierlayers can also be made of non-polymeric materials such as porous formsof metals, semiconductors, and ceramics. Representative examples ofpassive semiconductor barrier layer materials include nanoporous ormicroporous silicon membranes. Materials can be selected for use asbarrier layers to give a variety of perneabilities or degradation,dissolution, or disintegration rates. To obtain different delay times(time required for the barrier layer to become permeable and “expose”the reservoir contents) using polymeric embodiments, barrier layers canbe formed of different polymers, the same polymer with differentthicknesses, degrees of crosslinking, or an ultra-violet (UV) lightpolymerizable polymer. In the latter case, varying the exposure of thispolymer to UV light results in varying degrees of crosslinking and givesthe barrier layer different diffusion properties (i.e. permeabilities)or degradation, dissolution, or disintegration rates. Another way tocontrol the time at which the barrier layer becomes permeable is byusing one polymer, but varying the thickness of that polymer. Thickerfilms of some polymers result in a delay in the time to barrier layerpermeability. Any combination of polymer, degree of crosslinking, orpolymer thickness can be modified to obtain a specific delay time. Inone embodiment, the reservoir is covered by a degradable barrier layerthat is nearly impermeable to the molecules of interest. The time toinitiation of exposure of the reservoir contents will be limited by thetime necessary for the barrier layer material to degrade. In anotherembodiment, the barrier layer is non-degradable and is permeable tospecific molecules or types of energy (e.g., light) in the environment.The physical properties of the material used, its degree ofcrosslinking, its porosity, and its thickness will determine the timenecessary for the molecules or energy to diffuse or pass through thebarrier layer.

In active devices, the barrier layer can include any material that canbe disintegrated or permeabilized in response to an applied stimulus(e.g., electric field or current, magnetic field, change in pH, or bythermal, chemical, electrochemical, or mechanical means). In a preferredembodiment, the barrier layer is a thin metal (e.g., gold) membrane andis impermeable to the surrounding environment (e.g., body fluids oranother chloride containing solution). Based on the type of metal andthe surrounding environment, a particular electric potential (e.g.,+1.04 volts vs. a saturated calomel reference electrode) is applied tothe metal barrier layer. The metal barrier layer oxidizes and dissolvesby an electrochemical reaction, “exposing” the contents of the reservoirto the surrounding environment. In addition, materials that normallyform insoluble ions or oxidation products in response to an electricpotential can be used if, for example, local pH changes near the anodecause these oxidation products to become soluble. Examples of suitablebarrier layer materials include metals such as copper, gold, silver, andzinc, and some polymers, as described, for example, in Kwon et al.,Nature, 354:291-93 (1991); and Bae et al., ACS Symposium Series, 545:98-110 (1994). In another embodiment, the barrier layer is a polymerwith a melting point slightly above room temperature. When the localtemperature near the polymer barrier layer is increased above thepolymer's melting point by thin film resistors located near the barrierlayer, the barrier layer melts and exposes the contents of the reservoirto the surrounding environment.

Any combination of passive or active barrier layers can be present in asingle microchip device. Passive and active barrier layers can also becombined to form a multi-laminate or composite barrier layer. In onesuch embodiment, an impermeable, active barrier layer can be placed ontop of a permeable, passive barrier layer. When it is desired to exposethe contents of the reservoir to the surrounding environment, theimpermeable, active barrier layer is removed by the application of astimulus, such as an electric current. After the removal of the activebarrier layer, the passive layer still remains over the reservoir. Thepassive barrier layer is permeable to the molecules in the surroundingenvironment. However, the rate at which molecules pass through thepassive barrier layer was pre-determined during device fabrication bythe choice of the material used for the passive barrier layer, itsthickness, and its other physical and chemical properties.

E. Device Packaging, Control Circuitry, and Power Source

Active devices require actuation, which typically is done under thecontrol of a microprocessor. The microprocessor is programmed toinitiate the disintegration or permeabilization of the barrier layer inresponse to a variety of conditions, including a specific time, receiptof a signal from another device (for example by remote control orwireless methods), or detection of a particular condition using a sensorsuch as a biosensor.

Microelectronic device packages are typically made of an insulating ordielectric material such as aluminum oxide or silicon nitride. Low costpackages can also be made of plastics. Their purpose is to allow allcomponents of the device to be placed in close proximity and tofacilitate the interconnection of components to power sources and toeach other. For in vivo applications of the microchip device, the entirepackage, including all components (i.e. the device, the microprocessor,and the power source), are coated or encapsulated in a biocompatiblematerial such as poly(ethylene glycol) or polytetrafluoroethylene-likematerials. The materials requirements for in vitro applications aretypically less stringent and depend on the particular situation.

The control circuitry consists of a microprocessor, a timer, ademultiplexer, and an input source, for example, a memory source, asignal receiver, or a biosensor. Additional components can be added tothe system depending on the desired mode of barrier actuation (e.g.,thin film resistors for meltable barrier layers). The timer anddemultiplexer circuitry can be designed and incorporated directly ontothe surface of the microchip during electrode fabrication. The criteriafor selection of a microprocessor are small size, low power requirement,and the ability to translate the output from memory sources, signalreceivers, or biosensors into an address for the direction of powerthrough the demultiplexer to a specific reservoir on the microchipdevice (see, e.g., Ji, et al. IEEE J. Solid-State Circuits 27:433-43(1992)). Selection of a source of input to the microprocessor such asmemory sources, signal receivers, or biosensors depends on the microchipdevice's particular application and whether device operation ispreprogrammed, controlled by remote means, or controlled by feedbackfrom its environment (i.e., biofeedback).

The criteria for selection of a power source are small size, sufficientpower capacity, ability to be integrated with the control circuitry, theability to be recharged, and the length of time before recharging isnecessary. Batteries can be separately manufactured (i.e. off-the-shelf)or can be integrated with the microchip itself. Several lithium-based,rechargeable microbatteries are described in Jones & Akridge,“Development and performance of a rechargeable thin-film solid-statemicrobattery”, J. Power Sources, 54:63-67 (1995); and Bates et al., “Newamorphous thin-film lithium electrolyte and rechargeable microbattery”,IEEE 35^(th) International Power Sources Symposium, pp. 337-39 (1992).These batteries are typically only ten microns thick and occupy 1 cm² ofarea. One or more of these batteries can be incorporated directly ontothe microchip device. Binyamin, et al., J. Electrochem. Soc.,147.2780-83 (2000) describes work directed toward development of biofuelcells, which may provide a low power source suitable for the operationof the microchip devices described herein, as well as othermicroelectronic devices, in vivo.

II. Methods of Making the Microchip Devices

A. Fabrication of the Substrates with Reservoirs

Devices are manufactured using methods known in the art, reviewed forexample, by Wolf et al. (1986), Jaeger (1988), and Madou, Fundamentalsof Microfabrication (CRC Press 1997). The microchip devices can be madeusing the methods described below, alone or in combination with themethods described in U.S. Pat. Nos. 5,797,898 and 6,123,861, to Santini,et al.

In a preferred method of microchip manufacture, fabrication begins bydepositing and photolithographically patterning a material, typically aninsulating or dielectric material, onto the substrate to serve as anetch mask during reservoir etching. Typical insulating materials for useas a mask include silicon nitride, silicon dioxide, and some polymers,such as polyimide. In a preferred embodiment, a thin film (approximately1000-3000 Å) of low stress, silicon-rich nitride is deposited on bothsides of a silicon wafer in a Vertical Tube Reactor (VTR).Alternatively, a stoichiometric, polycrystalline silicon nitride (Si₃N₄)can be deposited by Low Pressure Chemical Vapor Deposition (LPCVD), oramorphous silicon nitride can be deposited by Plasma Enhanced ChemicalVapor Deposition (PECVD). Reservoirs are patterned into the siliconnitride film on one side of the wafer by ultraviolet photolithographyand either plasma etching or a chemical etch consisting of hotphosphoric acid or buffered hydrofluoric acid. The patterned siliconnitride serves as an etch mask for the chemical etching of the exposedsilicon by a concentrated potassium hydroxide solution (approximately20-40% KOH by weight at a temperature of 75-90° C.). Alternatively, thereservoirs can be etched into the substrate by dry etching techniquessuch as reactive ion etching, deep trench etching, or ion beam etching.Use of these microfabrication techniques allows the incorporation ofhundreds to thousands of reservoirs on a single microchip. The spacingbetween each reservoir depends on its particular application and whetherthe device is a passive or active device. Depending on the shape of thereservoirs and the sealing method used, the reservoirs of passive oractive devices may be as little as a few microns apart. Reservoirs canbe made in nearly any shape and depth, and need not pass completelythrough the substrate. In a preferred embodiment, the reservoirs areetched into a (100) oriented, silicon substrate by potassium hydroxide,in the shape of a square pyramid having side walls sloped at 54.7°, andpass completely through the substrate (approximately 300 to 600 μmthick) to the silicon nitride film on the other side of the substrate,forming a silicon nitride membrane. (Here, the silicon nitride filmserves as a potassium hydroxide etch stop.) The pyramidal shape allowseasy filling of the reservoirs with chemicals or devices through thelarge opening of the reservoir (approximately 500 μm by 500 μm for a 300μm thick wafer) on the patterned side of the substrate, exposure throughthe small opening of the reservoir (approximately 50 μm by 50 μm) on theother side of the substrate, and provides a large cavity inside thedevice for storing reacting components and secondary devices.

Multi-portion substrate devices can be formed simply by making two ormore individual substrate portions and then bonding them to one anotherwith the matching openings of the reservoir portions aligned. There aretwo main types of bonds that can be formed between substrate portions.The first are atomic-scale or molecular-scale bonds. These types ofbonds usually involve the interpenetration, intermixing, orinterdiffusion of atoms or molecules of one or both of the substrates atthe interface between the substrate materials. A preferred method ofthis type of substrate bonding for use primarily with silicon or glasssubstrates involves using heat and/or electric voltages to enable theinterdiffusion of material between the two substrates, causing amolecular-scale bond to form at the interface between silicon, glass,and other similar materials. This anodic bonding process is well knownto those skilled in the art. Another embodiment of this type of bondinginvolves melting and re-solidification of the top layer of one or bothsubstrates. The melted material intermixes and upon solidification, astrong bond is formed between the two substrates. In one embodiment,this kind of melting and re-solidification can be caused by the briefapplication of a solvent, such as methylene chloride, to the substrate,such as poly(methyl methacrylate) or PLEXIGLAS™. The second type ofbonding methods involves using a material other than the substratematerial to form the bond. A preferred embodiment of this type ofbonding includes the use of chemical adhesives, epoxies, and cements. Anembodiment that can be used with UV transparent substrate materialsinvolves UV curable epoxy. The UV curable epoxy is spread between thetwo substrate portions using a method such as spin coating, thereservoirs are aligned, and a UV light source is used to cross-link, orcure, the epoxy and bond the substrates together.

Alternatively, reservoirs can be formed using silicon-on-insulator (SOI)techniques, such as is described in Renard, J. Micromech. Microeng.10:245-49 (2000). SOI methods can be usefully adapted to form reservoirshaving complex reservoir shapes, for example, as shown in FIGS. 12A-C.SOI wafers behave essentially as two substrates that have been bonded onan atomic or molecular-scale before any reservoirs have been etched intoeither substrate. SOI substrates easily allow the reservoirs on eitherside of the insulator layer to be etched independently, enabling thereservoirs on either side of the insulator layer to have differentshapes. The reservoirs on either side of the insulator layer can then beconnected to make a single reservoir having a complex geometry byremoving the insulator layer between the two reservoirs using methodssuch as reactive ion etching, laser, ultrasound, or wet chemicaletching.

In other methods, the substrate is formed from polymer, ceramic, ormetal for example by compression molding powders or slurries of polymer,ceramic, metal, or combinations thereof. Other forming methods usefulwith these materials include injection molding, thermoforming, casting,machining, and other methods known to those skilled in the art.Substrates formed using these methods can be formed (e.g., molded) tohave the reservoirs or the reservoirs can be added in subsequent steps,such as by etching.

B. Fabrication of Passive Barrier Layers

In the fabrication of passive microchip devices, the barrier layermaterial is injected with a micro-syringe, printed with an inkjetprinter cartridge, or spin coated into a reservoir having the thinmembrane of insulating mask material still present over the smallopening of the reservoir. If injection or inkjet printing methods areused, barrier layer formation is complete after the material is injectedor printed into the reservoir and does not require further processing.If spin coating is used, the barrier layer material is planarized bymultiple spin coatings. The surface of the film is then etched by aplasma, an ion beam, or chemical etchant until the desired barrier layerthickness is obtained. After deposition of the barrier layer material,and possibly after reservoir filling, the insulating mask material isremoved, typically via dry or wet etching techniques. In a preferredembodiment the insulating material used is silicon nitride and thebarrier layer material is printed into the reservoir with an inkjetcartridge filled with a solution or suspension of the barrier layermaterial.

Barrier layers control the time at which secondary devices and/orreacting components are exposed to the surrounding environmentalcomponents or released from the reservoirs. Each barrier layer can be ofa different thickness or have different physical properties to vary thetime at which reservoir contents are exposed to the surrounding fluids.Injection, inkjet printing, and spin coating are preferred methods ofreservoir filling and any of these methods may be used to fillreservoirs, regardless of the reservoir's shape or size. However,injection and inkjet printing are the preferred methods of filling deep(greater than 10 μm) reservoirs or reservoirs with large openings(greater than 100 μm). For example, to obtain different barrier layerthicknesses using injection or inkjet printing, different amounts ofbarrier layer material are injected or printed directly into eachindividual reservoir. Spin coating is the preferred method of fillingshallow (less than 10 μm) reservoirs, reservoirs that do not passcompletely through the substrate, or reservoirs with small (less than100 μm) openings. Variation in barrier layer thickness or material byspin coating can be achieved by a repeated, step-wise process of spincoating, masking selected reservoirs, and etching. For example, to varybarrier layer thickness with spin coating, the barrier layer material isspin coated over the entire substrate. Spin coating is repeated, ifnecessary, until the material is nearly planarized. A mask material suchas photoresist is patterned to cover the barrier layer material in allthe reservoirs except one. Plasma, ion beam, or chemical etchants areused to etch the barrier layer material in the exposed reservoir to thedesired thickness. The photoresist is then removed from the substrate.The process is repeated as a new layer of photoresist is deposited andpatterned to cover the barrier layer material in all the reservoirsexcept one (the exposed reservoir is not the same one already etched toits desired thickness). Etching of the exposed barrier layer material inthis reservoir continues until the desired barrier layer thickness isobtained. This process of depositing and patterning a mask material suchas photoresist, etching, and mask removal can be repeated until eachreservoir has its own unique barrier layer thickness. The techniques,such as UV photolithography, and plasma or ion beam etching, are wellknown to those skilled in the field of microfabrication.

Although injection, inkjet printing and spin coating are the preferredmethods of barrier layer fabrication, it is understood that eachreservoir can be capped individually by capillary action, by pulling orpushing the material into the reservoir using a vacuum or other pressuregradient, by melting the material into the reservoir, by centrifugationand related processes, by manually packing solids into the reservoir, orby any combination of these or similar reservoir filling techniques.

C. Fabrication of Active Barrier Layers

In active devices, the barrier layer is located on, in, or covering eachreservoir. The active barrier layers consist of any material that can beremoved (e.g., disintegrated) or made permeable in response to anapplied stimulus (e.g., electric field or current, magnetic field,change in pH, or by thermal, photochemical, chemical, electrochemical,or mechanical means). Examples of active barrier layer materials includemetals such as copper, gold, silver, and zinc, and some polymers, asdescribed, for example, in Kwon et al., Nature, 354,291-93 (1991); andBae et al., ACS Symposium Series, 545: 98-110 (1994). Barrier layers andany related circuitry are deposited, patterned, and etched usingmicroelectronic and MEMS fabrication methods well known to those skilledin the art, reviewed, for example, by Wolf et al. (1986), Jaeger (1988),and Madou, Fundamentals of Microfabrication (CRC Press 1997). Inaddition, active barrier layers and associated circuitry can also beformed on the surface of microchip devices using microcontact printingand soft lithography methods, as described, for example, in Yan, et al.,J. Amer. Chem. Soc., 120:6179-80 (1998); Xia, et al., Adv. Mater.,8(12):1015-17 (1996); Gorman, et al., Chem. Mater., 7:52-59 (1995); Xia,et al., Annu. Rev. Mater. Sci., 28:153-84 (1998); and Xia, et al.,Angew. Chem. Int. Ed. 37:550-75 (1998).

In a preferred embodiment, the barrier layer is defined using a lift-offtechnique. Briefly, photoresist is patterned in the form of electrodeson the surface of the substrate having the reservoirs covered by thethin membrane of insulating or dielectric material. The photoresist isdeveloped such that the area directly over the covered opening of thereservoir is left uncovered by photoresist and is in the shape of ananode. A thin film of conductive material capable of dissolving intosolution or forming soluble ions or oxidation compounds upon theapplication of an electric potential is deposited over the entiresurface using deposition techniques such as chemical vapor deposition,electron or ion beam evaporation, sputtering, spin coating, and othertechniques known in the art. Exemplary materials include metals such ascopper, gold, silver, and zinc and some polymers, as disclosed by Kwonet al. (1991) and Bae et al. (1994). After film deposition, thephotoresist is stripped from the substrate. This removes the depositedfilm, except in those areas not covered by photoresist, which leavesconducting material on the surface of the substrate in the form ofelectrodes. An alternative method involves depositing the conductivematerial over the entire surface of the device, patterning photoresiston top of the conductive film using ultraviolet (UV) or infrared (IR)photolithography, so that the photoresist lies over the reservoirs inthe shape of anodes, and etching the unmasked conductive material usingplasma, ion beam, or chemical etching techniques. The photoresist isthen stripped, leaving conductive film anodes covering the reservoirs.Typical film thicknesses of the conductive material may range from 0.05to several microns. The anode serves as the active barrier layer and theplacement of the cathodes on the device is dependent upon the device'sapplication and method of electric potential control.

Following deposition of the electrodes, an insulating or dielectricmaterial such as silicon oxide (SiO_(X)) or silicon nitride (SiN_(X)) isdeposited over the entire surface of the device by methods such aschemical vapor deposition (CVD), electron or ion beam evaporation,sputtering, or spin coating. Photoresist is patterned on top of thedielectric to protect it from etching except on the cathodes and theportions of the anodes directly over each reservoir. The dielectricmaterial can be etched by plasma, ion beam, or chemical etchingtechniques. The purpose of this film is to protect the electrodes fromcorrosion, degradation, or dissolution in all areas where electrode filmremoval is not necessary for release.

The electrodes are positioned in such a way that when a suitableelectric potential is applied between an anode and a cathode, theunprotected (not covered by dielectric) portion of the anode barrierlayer oxidizes to form soluble compounds or ions that dissolves intosolution, compromising the barrier separating the reservoir contentsfrom the surrounding environment.

D. Removal of the Insulator Membrane (Reservoir Etch Stop)

The thin membrane of insulating or dielectric material covering thereservoir used as a mask and an etch stop during reservoir fabricationmust be removed from the active microchip device before filling thereservoir and from the passive microchip device (if the reservoirextends completely through the substrate) after filling the reservoir.The membrane may be removed in two ways. First, the membrane can beremoved by an ion beam or reactive ion plasma. In a preferredembodiment, the silicon nitride used as the insulating material can beremoved by a reactive ion plasma composed of oxygen and fluorinecontaining gases such as CHF₃, CF₄, or SF₆. Second, the membrane can beremoved by chemical etching. For example, buffered hydrofluoric acid(BHF or BOE) can be used to etch silicon dioxide and hot phosphoric acidcan be used to etch silicon nitride. If other materials are used as amembrane mask or etch stop, they can be removed using plasmacompositions or chemicals known to those skilled in the art of etching.

E. Reservoir Filling and Sealing

The chemicals and devices to be stored and protected within thereservoirs are inserted into one of the openings of each reservoir(e.g., the large opening of square pyramid-shaped reservoirs). Chemicalscan be inserted into the reservoir by injection, inkjet printing, orspin coating. Devices or device components can be fabricated inside ornear each reservoir, or can be fabricated away from the microchip andinserted into or placed near a reservoir during microchip and packagingassembly. Each reservoir can contain different chemicals, devices, ordevice components.

The distribution over the microchip of reservoirs filled with thechemicals or devices of interest can vary. For applications in medicaldiagnostics, for example, ink jet printing can be used to fill each rowof reservoirs on a microchip with different chemicals, each used todetect a particular analyte in solution. In another embodiment, eachreservoir is filled with a slurry of catalyst particles bymicroinjection. If desired, each reservoir can be filled with a catalystfor a different chemical reaction. In yet another embodiment, a solutionof a biological catalyst (i.e., enzyme) or a DNA marker molecule isinjected into a reservoir and allowed to dry, immobilizing the enzyme orthe DNA marker on the inner surface of the reservoir. Althoughinjection, inkjet printing, and spin coating are the preferred methodsof inserting chemicals into reservoirs, it is understood that eachreservoir can be filled individually by capillary action, by pulling orpushing the material into the reservoir using a vacuum or other pressuregradient, by melting the material into the reservoir, by centrifugationand related processes, by manually packing solids into the reservoir, orby any combination of these or similar reservoir filling techniques.

Each reservoir can also contain a different device or device component.Such devices can be fabricated directly in each reservoir. In oneembodiment, thin metal electrodes for use in a sensing application canbe fabricated onto the sidewalls of a pyramid-shaped reservoir usingphotolithography and electron beam evaporation. It is also possible tofabricate device components separately from the microchip and thenintegrate them with the microchip during the assembly process. In oneembodiment, a device used in an optical based assay (e.g., LED) isplaced into or near a reservoir during the assembly process. In anotherembodiment, a completely functional sensor (e.g., an ISFET or IonSelective Field Effect Transistor) is fabricated on another substrateportion. The substrate portion containing the sensor is aligned with thereservoir on the other substrate portion, and the two portions arebonded together, sealing the sensor inside the reservoir.

In preferred embodiments of both active and passive release devices, thereservoir openings used for chemical filling or device insertion (i.e.the openings opposite the barrier layer end) are sealed followingreservoir filling, using any of a variety of techniques known in theart. For example, sealing can be provided by compressing a thin flexiblefilm across the openings with a rigid backing plate. Alternatively, theopening can be sealed by applying a fluid material (e.g., an adhesive,wax, or polymer) that plugs the opening and hardens to form a seal. Inanother embodiment, a second substrate portion, e.g., of a seconddevice, can be bonded across the reservoirs openings.

F. Device Packaging, Control Circuitry, and Power Source

The openings through which the reservoirs of passive and active devicesare filled are sealed by compression, by wafer bonding, by a waterproofepoxy, or by another appropriate material impervious to the surroundingenvironment. For in vitro applications, the entire unit, except for theface of the device containing the reservoirs and barrier layers, isencased in a material appropriate for the system. For in vivoapplications, the unit is preferably encapsulated in a biocompatiblematerial such as poly(ethylene glycol) or polytetrafluoroethylene, or acase made of a biocompatible metal or ceramic.

The mechanism for exposing the reservoir contents of the device does notdepend on multiple parts fitted or glued together which must retract ordislodge. Exposing of the contents of each reservoir can be controlledby a preprogrammed microprocessor, by remote control, by a signal from abiosensor, or by any combination of these methods.

A microprocessor is used in conjunction with a source of memory such asprogrammable read only memory (PROM), a timer, a demultiplexer, and apower source such as a microbattery, as described, for example, by Joneset al. (1995) and Bates et al. (1992), or a biofuel cell, as describedby Binyamin, et al. (2000). A programmed sequence of events includingthe time a reservoir is to be opened and the location or address of thereservoir is stored into the PROM by the user. When the time forexposure or release has been reached as indicated by the timer, themicroprocessor sends a signal corresponding to the address (location) ofa particular reservoir to the demultiplexer. The demultiplexer routes aninput, such as an electric potential or current, to the reservoiraddressed by the microprocessor. A microbattery provides the power tooperate the microprocessor, PROM, and timer, and provides the electricpotential input that is directed to a particular reservoir by thedemultiplexer. The manufacture, size, and location of each of thesecomponents are dependent upon the requirements of a particularapplication. In a preferred embodiment, the memory, timer,microprocessor, and demultiplexer circuitry is integrated directly ontothe surface of the chip. The microbattery is attached to the other sideof the chip and is connected to the device circuitry by vias or thinwires. However, in some cases, it is possible to use separate,prefabricated, component chips for memory, timing, processing, anddemultiplexing. In a preferred embodiment, these components are attachedto the back side of the microchip device with the battery. In anotherpreferred embodiment, the component chips and battery are placed on thefront of or next to the microchip device, for example similar to how itis done in multi-chip modules (MCMs) and hybrid packages. The size andtype of prefabricated chips used depends on the overall dimensions ofthe microchip device and the number of reservoirs.

Activation of a particular reservoir by the application of an electricpotential or current can be controlled externally by remote control.Much of the circuitry used for remote control is the same as that usedin the preprogrammed method. A signal, such as radio frequency (RF)energy, microwaves, low power laser, or ultrasound, is sent to areceiver by an external source, for example, computers or ultrasoundgenerators. The signal is received by the microprocessor where it istranslated into a reservoir address. Power is then directed through thedemultiplexer to the reservoir having the appropriate address.

A biosensor can be integrated into or onto the microchip device todetect molecules in the surrounding fluids. When the concentration ofthe molecules reaches a certain level, the sensor sends a signal to themicroprocessor to activate one or more reservoirs. The microprocessordirects power through the demultiplexer to the particular reservoir(s).

III. Applications for the Microchip Devices

Passive and active microchip devices have numerous in vitro and in vivoapplications. The microchip devices can be used in a variety ofapplications in which it is desired to selectively expose molecules,devices, or a small volume (i.e. that of a reservoir) to anotherenvironment outside that volume. Applications include controlled orselective, on-demand sensing, for example to detect the presence orabsence of a type of molecule, to test for biological activity orreactivity of molecules exposed to the sensor, or to measure parameters,such as pH, temperature, reactivity with another molecule, opticalproperties (e.g., refractive index, color, or fluorescence),radioactivity, pressure, or electrical conductivity. In one embodiment,the sensor employs an optical fiber that can be used to sense changes inoptical properties in or near the reservoirs, changes which might occur,for example, due to a reaction in the reservoir or in the environmentadjacent the reservoir. In a related embodiment, the reservoir containsa scintillation fluid to aid in the (optical) detection of radioactivematerials.

In a preferred embodiment, the microchip device contains one or moresensors for use in glucose monitoring and insulin control. For example,one or more reservoirs could contain a sensor while other reservoirscontain insulin for release. Information from the sensor could be usedto actively control insulin release.

The microchip device can be used in vitro to selectively exposesecondary devices or device components, reacting components, or both tothe surrounding environment or components thereof. For some in vitroapplications, the microchip can release small, controlled amounts ofchemical reagents or other molecules into solutions or reaction mixturesat precisely controlled times and rates. In others, small devices suchas sensors can be protected from the surrounding environment until theyare needed. Analytical chemistry and medical diagnostics are examples offields where microchips having the ability to selectively exposechemicals and devices can be used. Such microchips can also be used invivo as delivery devices. The microchips can be implanted into apatient, either by surgical techniques or by injection, or can beswallowed. The microchips can provide delivery or sensing of manydifferent molecules and devices at varying rates and at varying times.Other microchips can be used to catalyze a particular reaction in vivo.For example, the catalyst (i.e. enzyme) can be protected in thereservoir from the surrounding environment until it is desired to exposethe enzyme and catalyze the reaction of interest.

The devices also can be used to isolate a reaction component, such asenzymes and other catalysts, for example in analytical chemistry ormedical diagnostics. For example, the reservoir can function as a packedbed reactor or immobilized enzyme reactor. In one embodiment, thedevices utilize osmotic pressure and/or swellable materials to open thereservoirs to permit molecules to enter or leave the reservoirs. Theseand other applications are detailed in the non-limiting embodimentsdescribed below, wherein it is understood that the number, geometry, andplacement of each reservoir, barrier layer, or other object (i.e.,heaters, electrodes, channels, etc.) in or near each reservoir can bemodified for a particular application. For simplicity, only one or tworeservoirs are shown in each Figure. However, it is understood that amicrochip component or device would contain at least two, and preferablymany more, reservoirs arrayed across a substrate.

A. Selective Sensing Device

In one embodiment, illustrated in FIGS. 2A-C, a sensor 22 for detectinga particular molecule is fabricated or placed at the bottom or on aninterior side of a reservoir 14 in substrate 12 of microchip device 10,having backing plate 16 and semi-permeable barrier layer 20. In FIG. 2A,barrier layer 20 covers the reservoir, allowing the passage of moleculeof interest “A” into or out of the reservoir 14 while restricting thepassage of other molecules or materials (e.g., cells or cellularcomponents) that may affect the sensing of the molecule of interest.When the microchip device is first placed into operation, thesemi-permeable barrier layer 20 can be directly in contact with thesurrounding environment, or it can be covered by another barrier layer18 that is impermeable to molecule “A”, as shown in FIG. 2B. In thelatter case, the impermeable barrier layer 18 prohibits the passage ofmaterial into or out of the reservoir 14 until the impermeable barrierlayer 18 is partially or completely removed, as shown in FIG. 2C, atwhich time the sensor 22 can then sense the presence or absence ofmolecule of interest “A”.

When the impermeable barrier layer can be partially or completelyremoved by the application of a stimulus (e.g., electric potential), theoperator or user of the microfabricated device has the ability toinitiate the operation of the sensor on demand. Such components ordevices could be useful in applications where sensor operation orperformance is diminished by exposure to a particular environment. Forexample, the performance of some implantable sensors has been observedto diminish as they become coated or “fouled” with cells, proteins, andother components found in vivo or in other operating environments.

B. Optical Sensing Device

In another embodiment, illustrated in FIGS. 3A-B, a miniature opticalfiber 24 is placed in or near a reservoir 14 disposed in substrateportions 12 a and 12 b of microchip device 10, having semi-permeablebarrier layer 20 and backing plate 16. Reservoir 14 contains one or moresubstances “X” that interact with one or more molecular or cellularcomponent of interest “A”, present in the environment around themicrochip device (outside the reservoir). As shown in FIG. 3B, thesubstance X inside the reservoir 14 is exposed by the partial removal ofan initially present barrier layer 18 to the environment containing themolecule or cellular component of interest “A”. Then an optical propertyof the substance inside reservoir 14 changes (X→X′) and is sensed viaoptical fiber 24. For example, the optical fiber 24 may be used toexpose the contents of the reservoir 14 to a light source, possibly of asingle wavelength. The optical fiber 24 also can have the ability todetect and measure changes in fluorescence, or some other opticalphenomenon. The excitation light source or detection source can beintegrated into the reservoir (FIG. 3A) or positioned externally from tothe reservoir (FIG. 3B). Such components or devices could be useful inmaking colorimetric diagnostic devices for the examination of bothbiological (e.g., proteins or DNA fragments) and non-biologicalsubstances.

C. Selective Sensor Device with Reagents

In another embodiment, illustrated in FIGS. 4-5, the reservoirs of themicrochip device contain a combination of reagents and sensors invarious configurations. For example, a reservoir containing one or moresensors can be filled with one or more reagents or other chemicalsrequired for conducting a particular assay.

FIG. 4A shows microchip device 10 having reservoir 14 that containssensor 22 and chemical reagent “S”, with reservoir 14 covered by barrierlayer 18. The barrier layer 18 isolates sensor 22 and chemical reagent“S” from the environment outside the reservoir. The environment containsor potentially contains molecule of interest “R”. As shown in FIG. 4B,when it is desired to activate the sensors, the barrier layer 18 is atleast partially removed, to permit the molecule of interest “R” to reactwith chemical reagent “S” to produce product T, which is sensed bysensor 22. The chemical reagent “S”, which is necessary for the assaymay remain in the reservoir 14 after the barrier layer is removed, ormay slowly pass out of the reservoir 14 while the molecule of interest“R” enters the reservoir 14. In other words, it is not critical whetherthe assay reaction occurs inside or just outside of the reservoir, solong as the reaction product can be sensed by the sensor.

In a variation of this embodiment, illustrated in FIG. 5A, microchipdevice 30 includes sensor 22 in a first reservoir 14 a and chemicalreagent “S” in one or more neighboring reservoirs 14 b, covered bybarrier layers 18 a and 18 b, respectively. As shown in FIG. 5B, sensingis initiated by removing barrier layers 18 a and 18 b to open reservoirs14 a and 14 b, which exposes sensor 22 and chemical reagent “S” to theenvironment outside the reservoirs, which includes molecule of interest“R”. Chemical reagent “S” passes out of reservoir 14 b, reacts moleculeof interest “R” to produce product T, which then is sensed by sensor 22in reservoir 14 a.

Alternatively, in certain applications, the sensor need not be locatedwithin a reservoir. For example, as shown in FIG. 6, microchip device 40includes reservoir 14 disposed in substrate 12 and containing chemicalreagent “S”. Sensor 22 is mounted on an exterior surface of substrate12. Again sensing is initiated by removing barrier layer 18 (shown inpartially removed form), thereby permitting chemical reagent “S” to exitreservoir 14 and react with molecule of interest “R” to produce productT, which then is sensed by sensor 22. It is evident that the assay inthese examples cannot be initiated until the barrier layer isdisintegrated or permeabilized and the sensor, reagents, and molecule ofinterest are no longer isolated from one another.

D. Control of Sensor Devices

In one embodiment, illustrated in FIG. 7, a microchip device 32 containstwo reservoirs, 14 a and 14 b, with each containing two sensors:reference sensor 26 and sensor 28. Reference sensor 26 is used to checkthe operation of sensor 28 in each reservoir. A microprocessor 36,powered by power source 38, can be programmed to continuously compare,using comparison units 34 a or 34 b, (e.g., voltmeters or otherinstrumentation), the operation of sensor 28 to the reference sensor 26in reservoir 14 a or 14 b. If for example sensor 28 in reservoir 14 a isnot operating properly, a signal can be sent back to the microprocessor36. The microprocessor 36, in turn, can activate reservoir 14 b andexpose the new pair of electrodes (i.e. sensor 28 and reference sensor26 in reservoir 14 b. In addition, the microprocessor 36 can send asignal to a transmitter 37 to notify a remotely located computer 39 thatonly one good sensor remains, or to signal other operationalinformation. While the Figure shows the reservoirs as open, it isunderstood that the one or more of the reservoirs can be provided in aninitially closed state, that is covered by a barrier layer untilexposure is desired.

E. Packed Bed Reactor

In another embodiment, the microchip device serves as a packed bedreactor, an example of which is illustrated in FIGS. 8A and 8B. Forexample, microchip device 50 includes a reservoir disposed in substrate12 and filled with catalyst 52. Catalyst 52 can be any catalyticmaterial or can be an inert, porous support coated with the catalyticmaterial. The reservoir is covered by barrier layer 18, which prohibitsor restricts the passage of reactants “A” to the catalyst or productsaway from the catalyst, as shown in FIG. 8A. Complete or partialremoval, or permeabilization, of the barrier layer 18 exposes catalyst52 to the environment outside of the reservoir and allows reactants “A”to contact the catalyst 52 and react to form product B, as shown in FIG.5B.

Microchip device 50 optionally is provided with reaction controlcomponent 54 positioned within the reservoir. Examples of these reactioncontrol components include resistive heaters and polarizable electrodes.These control components can be mounted in (e.g., on a bottom or sideinterior surface) or near the reservoir to assist in controlling therate of the reaction (A→B). FIGS. 8C and 8D show are top views of aresistive heater and a polarized electrode, respectively, located on thebottom of reservoir.

It is understood that a permeable or semi-permeable barrier layer alsocan be used in addition to or in place of an impermeable barrier layerto limit or control the types of molecules allowed to contact thecatalyst.

These microchip device reactors may be particularly useful inapplications where prolonged exposure of the catalyst to the environmentresults in decreased performance of the catalyst due to “fouling” orcoating of the catalyst surface or due to chemical degradation of thecatalyst, because these devices would enable many discrete quantities ofcatalyst to be contained in one small device, with each quantityavailable independently when needed. For example, if the catalyst of afirst reservoir becomes fouled, then a second reservoir can be opened toexpose fresh catalyst, and repeated for any number of reservoirs.Furthermore, different catalysts can be provided in different reservoirsof a single device, thereby enhancing the range of reactions that can becatalyzed with a single device.

F. Immobilized Enzyme Reactor

In still another embodiment, the reservoirs of the microchip device areprovided with an immobilized enzyme. For example, as illustrated inFIGS. 9A-B, microchip device 60 includes reservoir 14 disposed insubstrate 12 and covered by barrier layer 18. An enzyme 62 isimmobilized on one or more of the surfaces inside reservoir 14. Barrierlayer 18 covers the reservoir to isolate the enzyme 62 from theenvironment which includes reactant “A”. As illustrated in FIG. 9B,complete or partial removal of barrier layer 18 exposes immobilizedenzyme 62 to reactant “A”, which reacts to form product “B”.

Alternatively or in addition, one or more microorganisms (e.g., yeast,pyruvate) can be coated or immobilized on surfaces inside or near areservoir, For example, the microorganism may react with or catalyze areaction involving a molecular species that is undetectable by thesensor until reacted the microorganism to produce a second, detectablemolecular species.

It is understood that a permeable or semi-permeable barrier layer alsocan be used in addition to or in place of an impermeable barrier layerto limit or control the types of molecules allowed to contact theimmobilized enzyme. These microchip reactor devices can be useful inapplications where a highly selective enzyme is required, but thestability of the enzyme is decreased when exposed to a particularenvironment for prolonged periods of time.

G. Use of Osmotic Pressure and/or Swellable Materials

In one embodiment, reservoirs are opened by rupture of one or morebarrier layers initially covering the reservoirs. In one variation ofthis embodiment, rupture is initiated by employing osmotic pressure,water swellable materials, or combinations thereof. For example, areservoir in a microchip device can be filled or coated with a material(for example, a salt) that causes an osmotic pressure to develop whenexposed to materials from the environment outside or near the reservoir.Depending on the design of the reservoirs, the osmotic pressuregenerating materials selected, and the placement of the osmotic pressuregenerating materials in or near the reservoir, the osmotic pressure thatwould develop can be used to either pull material into the reservoir orexpel material from the reservoir.

One example of the use of osmotic pressure in microchip components ordevices, illustrated in FIG. 10, involves using the osmotic pressuregenerated in a reservoir to eject a chemical from the reservoir or froma neighboring reservoir. In FIG. 10, microchip device 70 includessubstrate 76, first reservoir 72 containing chemicals to be released 74,and second reservoir 71 containing a concentrated solution of ionicspecies “A” (the osmotic pressure generating material). The devicefurther includes semi-permeable barrier layer 73, through which water orother solvent for “A” can pass, and rupturable, impermeable barrierlayer 75. Semi-permeable barrier layer 73 optionally can be covered byanother impermeable barrier layer (not shown) that can be selectivelyremoved to expose barrier layer 73 to the surrounding environment. FIG.10 shows water permeating barrier layer 73 due to osmotic forces. Morespecifically, the concentration of ionic species “A” in reservoir 71 isgreater that the concentration of ionic species “A” in the aqueousenvironment, thereby driving water through barrier layer 73 to equalizethe concentration of species “A”. This increased quantity of water inreservoir 71 increases the pressure in reservoirs 71 and 72, until thepressure causes impermeable barrier layer 75 to rupture and releasechemicals 74. Reservoirs 71 and 72 may be separated by a flexible,fluid-tight membrane or any other means which allows a change in thepressure of one reservoir to affect the pressure in the other, but whichmaintains the separation of the contents of each reservoir.

In another variation of this embodiment, an osmotic pressure generatingmaterial and a chemical that is meant to be ejected or released from thereservoir are placed in the same reservoir. An example is illustrated inFIGS. 11A-B, which shows microchip device 80 including reservoir 84disposed in substrate 82 and covered by rupturable, semi-permeablebarrier layer 90. Reservoir 84 is filled with chemical to be released 86and osmotic pressure generating material 85. Filled reservoir 84initially is covered with an impermeable barrier layer 88 to keep thesurrounding solution from entering the reservoir before release from thereservoir is desired. When release is desired, a stimulus is applied tothe impermeable barrier layer 88 long enough to expose semi-permeablebarrier layer 90 and render it permeable to a solution outside of thereservoir 84, as shown in FIG. 11A. The solution (shown as H₂O) thenpasses through the barrier layer due to the osmotic pressure difference(i.e. driving force) between the environment inside and outside ofreservoir 84, for example due to different ion or salt concentrations.The pressure in reservoir 84 increases due to the flow of solution intothe reservoir 84 until the increased pressure causes the semi-permeablebarrier layer 90 and the remainder of impermeable barrier layer 88 torupture, thus causing the contents of the reservoir, chemicals 86 andosmotic pressure generating material 85, to be released into thesurrounding solution as illustrated in FIG. 11B.

In an alternative but similar embodiment, a swellable material can usedin place of the osmotic pressure generating material in the reservoir.The swellable material, such as a swellable polymer, will swell orexpand when exposed to a particular solution. The reservoir volume,barrier layer material and thickness, and swellable material type andvolume, can be selected to provide a system in which the swelling of theswellable material causes the barrier layer to rupture in much the sameway that the buildup of solution in the reservoir due to osmoticpressure caused the barrier layer to rupture in the preceding example.Various combinations of semi-permeable and impermeable barrier layerscan be used depending on the particular application and microchip devicedesign.

Publications cited herein and the materials for which they are cited arespecifically incorporated by reference. Modifications and variations ofthe methods and devices described herein will be obvious to thoseskilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe appended claims.

1. A method for making a reservoir-based sensor device, comprising:fabricating a first substrate portion which comprises an upper surface,an opposed lower surface, a plurality of through holes definingreservoirs between said upper surface and said lower surface, and aplurality of reservoir caps closing off one end of said through holes atsaid upper surface; fabricating a plurality of sensors on an uppersurface of a second substrate portion; aligning the first and secondsubstrate portions such that each of said plurality of sensors is inalignment with each of said plurality of reservoirs; and bondingtogether the lower surface of said first substrate portion and the uppersurface of said second substrate portion to seal each of said sensorsinside the reservoir with which it is aligned, wherein the reservoircaps each comprise a metal membrane operable to be selectivelydisintegrated to open one end of the through hole closed off by thereservoir cap.
 2. The method of claim 1, wherein the sensors arebiosensors.
 3. The method of claim 1, wherein the first and secondsubstrate portions comprise silicon, glass, or a combination thereof 4.The method of claim 1, wherein the bonding comprises a molecular-scalebond at the interface between the materials forming the first and secondsubstrate portions.
 5. The method of claim 1, wherein the bondingcomprises melting and re-solidification of the material forming theinterfacing surface of one or both of the first and second substrateportions.
 6. The method of claim 1, wherein the bonding comprises theuse of a chemical adhesive, epoxy, or cement.
 7. The method of claim 1,wherein the metal membrane comprises gold.
 8. The method of claim 1,wherein the selective disintegration of the metal membrane comprises aphase change in the metal membrane by application of an electric fieldor current.
 9. The method of claim 1, wherein the step of fabrication ofthe first substrate portion comprises fabricating electrical circuitryon the upper surface of the first substrate portion, wherein theelectrical circuitry is used in the selective disintegration of themetal membrane.
 10. The method of claim 9, wherein the electricalcircuitry comprises at least one electrode tat is connected to thereservoir cap on the upper surface of the first substrate portion. 11.The method of claim 9, wherein the electrical circuitry is fabricated bya deposition, patterning, and etching process; by microcontact printing;or by soft lithography methods.
 12. A method for making an implantablereservoir-based sensor device, comprising: fabricating a first substrateportion which comprises an upper surface, an opposed lower surface, aplurality of through holes defining reservoirs between said uppersurface and said lower surface, and a plurality of metal reservoir capsclosing off one end of said through holes at said upper surface;fabricating a plurality of biosensors on an upper surface of a secondsubstrate portion, the biosensors comprising an enzyme or antibody;aligning the first and second substrate portions such that each of saidplurality of biosensors is in alignment with each of said plurality ofreservoirs; bonding together the lower surface of said first substrateportion and the upper surface of said second substrate portion to sealeach of said biosensors inside the reservoir with which it is aligned;and packaging the bonded first and second substrate portions togetherwith control circuitry and a power source into a package suitable forimplantation into a patient.
 13. The method of claim 12, wherein themetal reservoir caps are operable to be selectively disintegrated toopen one end of the though hole closed off by the reservoir cap.
 14. Themethod of claim 13, wherein the control circuitry and power source areoperable to induce a phase change in the reservoir caps.
 15. The methodof claim 12, wherein the metal reservoir caps comprise gold.